Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis

We're a lab that does volume EM structural work to say how do platelets activate, how do they aggregate, how do they form a thrombus that causes bleeding cessation, and how, in the case of overstimulation, they bring about occlusive clotting? In doing this, the premise is that if we know the structure, we can then better target drugs and bring about better outcomes. We use mouse models in our research, as many researchers do, and we all have the challenge of establishing how our findings in mice, and in our case, in the arteries and veins in mice, carry over to the mechanisms in the much larger arteries and veins in humans.

We all hope that the principles are the same in mice and humans. If you take our work and using volume EM approaches, the big things that we've contributed are one to say that platelets are not just suicide bombers. They retain granules.

They do things in response to signaling. They have stable states, and these stable states are part of a normal thrombus, and they're also part of an occlusive clot. Two, we've brought the sense that there is really structure to the thrombus that forms.

It's not just simply a pile of bricks, but rather it's an ordered structure in which platelets are ordered with respect to their activation, et cetera. And three, we've brought the sense that one should be able to apply and understand from structure what needs to be either stimulated to get a good outcome or what needs to be suppressed to give a outcome that will protect people from bad outcomes. We collect sequential images at near-nanometer scale over near-millimeter distances.

We assemble those into the single montage image. Hence, we can observe any given spot within the image at a scale of near-nanometers to near-millimeter, which is sufficient to place individual features inside of the whole thrombus. Researchers now have a powerful tool and they can ask the new question, how do coagulation cascades and cellular signals or drugs act spatially at local to global levels in thrombi?

To begin, with a pair of scissors and blunt forceps, lift and cut the skin of an anesthetized mouse over the right jugular vein. Cut a round window in the skin, large enough to expose the jugular vein and some of the surrounding tissue. Gently push aside the adipose tissue, lymph nodes, and connective tissue using the blunt dissection technique.

Next, fill a 20 milliliter syringe with normal saline warmed to 37 degrees Celsius. Load the syringe into the syringe infusion pump, then attach a 27-gauge butterfly needle and associated tubing to the syringe. Set the syringe pump to a flow rate of 0.5 milliliters per minute to wash away blood from the punctured vein or artery.

Position the stream of saline a few milliliters to the side of the puncture site and not directly over it. Once the jugular vein has been cleaned, start the timer. Obtain 30 gauge and 33 gauge hypodermic needles for the jugular vein and the femoral artery respectively.

Turn the bevel to face upwards, then position the needle at a 25-degree angle over the jugular vein or femoral artery. After noting the time, quickly puncture the top of the vessel. Note the time taken for the bleeding to stop completely.

Wait for the formation of a thrombus before starting perfusion fixation. Once perfusion has been completed, dissect the part of the jugular vein with the puncture site and thrombus. Use a pair of sharp scissors to make a diagonal cut across the distal end of the vessel segment and make a straight cut across the proximal end of the vessel segment.

Next, place the fixed tissue at four degrees Celsius for 24 hours. The next day, pour sodium cacodylate buffer solution into a 35 millimeter culture dish containing a five millimeter thick silicone mat. Carefully transfer the tissue into the buffer, ensuring that it is submerged in the buffer.

With stainless steel minutien pins and a pair of fine forceps, carefully pin each end of the vessel to the mat. Carefully clean the jugular vein. With a pair of microscissors, cut the vessel wall opposite the puncture site lengthwise.

Gently open the vessel, then pin it to the silicone mat with the intraluminal side facing up. Remove the buffer solution from the dish, then add 1%paraformaldehyde to submerge the vessel. Immediately place the lid on the dish to prevent drying out.